Translational control perks up.
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Amino Acids↗
Biomedical subjects
B R Williams
Publications and source records attributed to B R Williams.
Cell cycle regulation of the double stranded RNA activated protein kinase, PKR.
The interferon (IFN)-induced, double stranded RNA (dsRNA)-activated serine/threonine kinase, PKR, is a potent negative regulator of cell growth when overexpressed in yeast or mammalian cells. To determine whether endogenous PKR plays a role in cell growth control, we have investigated the regulation of PKR levels and activity during the cell cycle in human glioblastoma T98G cells. The steady-state level of PKR mRNA in T98G cells was highest in growth arrested cells, dropped sharply within 3 h of serum stimulation then gradually increased as cells progressed through G1, reaching a plateau in early S phase. PKR protein level increased following serum stimulation reaching a peak at the G2+M boundary and declining thereafter. In contrast, PKR kinase activity exhibited two peaks, in early G1 and at the G1/S boundary, declining sharply in early S phase. Thus, the activity profile did not follow the protein profile indicating a tight regulation of PKR at the level of activity. In T98G cells expressing the catalytically inactive PKRK296R the dsRNA-induced activation of NF-kappaB and IRF-1 was suppressed and the mutant cells exhibited resistance to stress induced apoptosis. Cell cycle distribution analysis showed that the mutant expressing cells exhibited longer G1 phase and fewer cells engaged in S phase. Furthermore, early passage mouse embryo fibroblasts derived from PKR knockout mice grew more slowly compared with the control cells. Taken together these results suggest that PKR may play a role in cell cycle progression.
Amino Acid Sequence↗
The DNA-binding subunit p140 of replication factor C is upregulated in cycling cells and associates with G1 phase cell cycle regulatory proteins.
The DNA-binding subunit of replication factor C (RFCp140) plays an important role in both DNA replication and DNA repair. The mechanisms regulating activation of RFCp140 thereby controlling replication and cellular proliferation are largely unknown. We analyzed protein expression of RFCp140 during cell cycle progression and investigated the association of RFCp140 with cell cycle regulatory proteins in cell lines of various tissue origin and in primary hematopoietic cells. Western and Northern blot analyses of RFCp140 from synchronized cells showed downregulation of RFCp140 when cells enter a G0-like quiescent state and upregulation of RFCp140 in cycling cells. Translocation from the cytoplasmic compartment to the nucleus did not account for the significant increase in RFCp140 protein levels observed in cycling cells. To investigate a potential association of RFCp140 with cell cycle regulatory proteins coimmunoprecipitation assays were performed. These studies demonstrated specific binding of RFCp140 to cdk4-kinase in hematopoietic and fibroblast cell lines. Additional coimmunoprecipitation studies revealed specific association of RFCp140 with cyclin D1, p21, proliferating cell nuclear antigen, and retinoblastoma protein. These findings link DNA replication and repair factor RFCp140 to G1 phase cell cycle regulatory elements critically involved in cell cycle control.
3T3 Cells↗
Development of inhibitory control across the life span.
The stop-signal procedure was used to examine the development of inhibitory control. A group of 275 participants, 6 to 81 years of age, performed a visual choice reaction time (go) task and attempted to inhibit their responses to the go task when they heard a stop signal. Reaction times to the stop and go signals were used to assess performance in inhibition and response execution, respectively. Results indicated the speed of stopping becomes faster with increasing age throughout childhood, with limited evidence of slowing across adulthood. By contrast, strong evidence was obtained for age-related speeding of go-signal reaction time throughout childhood, followed by marked slowing throughout adulthood. Hierarchical regression confirmed that the age-related change in inhibitory control could not be explained by general speeding or slowing of responses. Findings are discussed in regard to the contrast between the development of inhibition and response execution and the utility of the stop-signal procedure.
Adolescent↗
Inhibition of double-stranded RNA- and tumor necrosis factor alpha-mediated apoptosis by tetratricopeptide repeat protein and cochaperone P58(IPK).
P58(IPK) is a tetratricopeptide repeat-containing cochaperone that is involved in stress-activated cellular pathways and that inhibits the activity of protein kinase PKR, a primary mediator of the antiviral and antiproliferative properties of interferon. To gain better insight into the molecular actions of P58(IPK), we generated NIH 3T3 cell lines expressing either wild-type P58(IPK) or a P58(IPK) deletion mutant, DeltaTPR6, that does not bind to or inhibit PKR. When treated with double-stranded RNA (dsRNA), DeltaTPR6-expressing cells exhibited a significant increase in eukaryotic initiation factor 2alpha phosphorylation and NF-kappaB activation, indicating a functional PKR. In contrast, both of these PKR-dependent events were blocked by the overexpression of wild-type P58(IPK). In addition, the P58(IPK) cell line, but not the DeltaTPR6 cell line, was resistant to dsRNA-induced apoptosis. Together, these findings demonstrate that P58(IPK) regulates dsRNA signaling pathways by inhibiting multiple PKR-dependent functions. In contrast, both the P58(IPK) and DeltaTPR6 cell lines were resistant to tumor necrosis factor alpha-induced apoptosis, suggesting that P58(IPK) may function as a more general suppressor of programmed cell death independently of its PKR-inhibitory properties. In accordance with this hypothesis, although PKR remained active in DeltaTPR6-expressing cells, the DeltaTPR6 cell line displayed a transformed phenotype and was tumorigenic in nude mice. Thus, the antiapoptotic function of P58(IPK) may be an important factor in its ability to malignantly transform cells.
3T3 Cells↗
Medication use in residential care facilities for the elderly.
OBJECTIVE: To describe medication use by residents of residential care facilities for the elderly (RCFEs). DESIGN: A cross-sectional survey of medication use. SETTING: Licensed, private RCFEs recruited from a roster of all licensed RCFEs in the Los Angeles area. SUBJECTS: Residents who were > or =60 years of age and whose medications were centrally stored in the facility. MEASURES: Age, gender, race, health insurance coverage, dietary restrictions, ambulation status, medical diagnoses, and medication profile. RESULTS: A total of 818 residents were surveyed. Residents were primarily white women who were >80 years. The average number of medications per resident was five; 94% of the sample took at least one medication. Cardiovascular drugs, central nervous system drugs, analgesics, diuretics, and potassium supplements were most commonly used. Use of multiple drugs within a therapeutic class was also common, with means ranging from 1.46 to 1.81 per resident for the most commonly prescribed classes. Diagnoses supporting the use of many medications were not documented in the residents' health records. CONCLUSIONS: This RCFE sample was medically frail and took many medications. The frequent use of cardiovascular medication reflected the prevalence of cardiac disease in the elderly. The frequency of psychotropic drug use without a corresponding indication suggested prescribing for symptoms rather than documented medical conditions. Lack of recorded diagnoses limited the ability to evaluate drug therapy. Improved record keeping; periodic medication review; and resident, staff, and prescriber education are necessary to ensure appropriate medication use in this setting.
Aged↗
Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays.
The pleiotropic activities of interferons (IFNs) are mediated primarily through the transcriptional regulation of many downstream effector genes. The mRNA profiles from IFN-alpha, -beta, or -gamma treatments of the human fibrosarcoma cell line, HT1080, were determined by using oligonucleotide arrays with probe sets corresponding to more than 6,800 human genes. Among these were transcripts for known IFN-stimulated genes (ISGs), the expression of which were consistent with previous studies in which the particular ISG was characterized as responsive to either Type I (alpha, beta) or Type II (gamma) IFNs, or both. Importantly, many novel IFN-stimulated genes were identified that were diverse in their known biological functions. For instance, several novel ISGs were identified that are implicated in apoptosis (including RAP46/Bag-1, phospholipid scramblase, and hypoxia inducible factor-1alpha). Furthermore, several IFN-repressed genes also were identified. These results demonstrate the usefulness of oligonucleotide arrays in monitoring mammalian gene expression on a broad and unprecedented scale. In particular, these findings provide insights into the basic mechanisms of IFN actions and ultimately may contribute to better therapeutic uses for IFNs.
Apoptosis↗
Protein-tyrosine phosphatase Shp-1 is a negative regulator of IL-4- and IL-13-dependent signal transduction.
Binding of interleukin (IL)-4 to its transmembrane receptor results in the Jak-mediated tyrosine phosphorylation of a number of protein components of the IL-4 signaling cascade, including Jak1, Jak2, Jak3, Tyk2, IL-4Ralpha, IRS-1, IRS-2, and Stat6 in appropriate cell types. However, the protein-tyrosine phosphatases (PTPs) that dephosphorylate these proteins and terminate signaling remained unidentified. We have noted that IL-4-dependent activation of Stat6 is sustained longer in fibroblasts than in lymphoid cells. Because Shp-1, an SH2 domain-containing PTP, is expressed primarily in hematopoietic cells, we examined whether Shp-1 activity could regulate IL-4-dependent cell signaling. Expression of an Shp-1 transgene in NIH 3T3 cells markedly reduces both IL-4-dependent Stat6 activation and Stat6-mediated transcription of IL-4-responsive genes. In accord with this, IL-4 treatment of bone marrow-derived macrophages from viable motheaten mice that express substantially reduced levels of Shp-1 activity show remarkably enhanced activation of Stat6. In addition, Stat6 activation by IL-4 is significantly enhanced in pre-B cells derived from motheaten (Shp-1 null mutant) mice compared with normal pre-B cells derived from control animals. These data clearly implicate Shp-1 in the negative regulation of the IL-4/IL-13-activated Jak-Stat pathway.
3T3 Cells↗
Constitutive expression of the Wilms tumor suppressor gene (WT1) in renal cell carcinoma.
The expression of the Wilms tumor suppressor gene WT1 is largely restricted to elements of the developing urogenital system. In the fetal kidney, WT1 transcripts are present at low levels in the condensing mesenchyme and at much higher levels in differentiating glomerular epithelium and are not detected in other mesenchymal-derived epithelial structures such as the proximal and distal tubules. However, WT1 expression is observed in tubule-like elements found in some Wilms tumors. As renal cell carcinoma (RCC) of the clear cell type is one of the most prevalent adult tumors of the kidney, and is thought to originate from the epithelial cells of the proximal tubules, we studied WT1 expression in RCCs. Despite the absence of WT1 in normal primary epithelial cells derived from proximal tubules, RCC tumors and tumor-derived cell lines expressed WT1 RNA. Immunocytochemical analyses of tumor cryosections showed widespread expression throughout the poorly differentiated epithelial components of the tumor. Immunoblots of RCC samples detected a normal size WT I protein and reciprocal antibody immunoprecipitations of RCC cell extracts indicated that WT I interacts with p53 as has been demonstrated for normal human fetal kidney. The aberrant expression of functional WT1 in RCC may represent a reversion to a more de-differentiated phenotype and may contribute to the tumorigenic phenotype by inappropriately activating or repressing genes involved in growth regulation.
Alternative Splicing↗
Subcellular localization of the von Hippel-Lindau disease gene product is cell cycle-dependent.
The von Hippel-Lindau gene product (pVHL) interacts with and inhibits the cellular transcription factor elongin. However, the subcellular localization of pVHL has remained uncertain. Naturally occurring pVHL mutants which fail to interact with elongin have been described in patients with VHL disease or sporadic renal cell carcinoma (RCC). Here, we have examined the cellular expression pattern of endogenous pVHL in different RCC cell lines by immunocytochemistry and confocal microscopy. Both anti-N-terminal and anti-C-terminal pVHL antibodies were able to recognize endogenous wild-type pVHL expressed by the RCC cells studied. A C-terminal truncated VHL mutant expressed by RCC cell line A498 was detected only by the N-terminal antibody but not by the C-terminal antibody as expected. The overall staining patterns of these cell lines are similar, with a predominant nuclear speckled pattern and a moderate cytoplasmic staining in subconfluent cell cultures. Interestingly, when cells reached confluency, more prominent nuclear staining with little or no cytoplasmic expression was observed. By using double labeling with anti-VHL and anti-bromodeoxyuridine (BrdU) antibodies and cell cycle analyses, we found that in the G1/G0-phase, pVHL was localized exclusively in the nucleus associated with distinctive large subnuclear structures, whereas the majority of the cells in S-phase of the cell cycle also showed a diffuse cytoplasmic staining. Our results indicate that subcellular localization of pVHL is regulated in a cell cycle-dependent manner.
Carcinoma, Renal Cell↗
Structure of the double-stranded RNA-binding domain of the protein kinase PKR reveals the molecular basis of its dsRNA-mediated activation.
Protein kinase PKR is an interferon-induced enzyme that plays a key role in the control of viral infections and cellular homeostasis. Compared with other known kinases, PKR is activated by a distinct mechanism that involves double-stranded RNA (dsRNA) binding in its N-terminal region in an RNA sequence-independent fashion. We report here the solution structure of the 20 kDa dsRNA-binding domain (dsRBD) of human PKR, which provides the first three-dimensional insight into the mechanism of its dsRNA-mediated activation. The structure of dsRBD exhibits a dumb-bell shape comprising two tandem linked dsRNA-binding motifs (dsRBMs) both with an alpha-beta-beta-beta-alpha fold. The structure, combined with previous mutational and biochemical data, reveals a highly conserved RNA-binding site on each dsRBM and suggests a novel mode of protein-RNA recognition. The central linker is highly flexible, which may enable the two dsRBMs to wrap around the RNA duplex for cooperative and high-affinity binding, leading to the overall change of PKR conformation and its activation.
Amino Acid Sequence↗
Loss of heterozygosity at chromosome 11p15 in Wilms tumors: identification of two independent regions.
Loss of heterozygosity (LOH) on the short arm of chromosome 11 is the most frequent genetic alteration in Wilms tumors, indicating that one or more tumor suppressor genes that map to this chromosomal region are involved in the development of the disease. The WT1 gene located on 11p13 has been characterized but mutations in this gene occur in only about 10% of Wilms tumors. A second locus (WT2) at chromosome 11p15 has also been described in Wilms tumors but thus far efforts to clone the WT2 gene(s) have been frustrated by the large size (approximately 10 Mb) of this region. Using a high-density marker LOH analysis of 11p15.5-15.4, we have refined the location of a Wilms tumor suppressor gene between the markers D11S1318-D11S1288 (approximately 800 kb) within 11p15.5. We have also identified a second, novel region of LOH that spans the markers D11S1338-D11S1323 (approximately 336 kb) at 11p15.5-p115.4. Thus a second distinct locus, in addition to the previously defined WT2, on chromosome 11p15.5, appears to play a role in the development of Wilms tumors.
Adolescent↗
Biological properties of recombinant alpha-interferons: 40th anniversary of the discovery of interferons.
IFNs were first described as potent antiviral agents 40 years ago, and recombinant IFN-alpha2a and IFN-alpha2b were approved for the treatment of hairy cell leukemia just 11 years ago. Today, alpha-IFNs are approved worldwide for the treatment of a variety of malignancies and virologic diseases. Although the exact mechanism of action of IFN-alpha in the treatment of such diseases is not fully understood, many advances have been made in the characterization of the physicochemical and diverse biological properties of this highly pleiotropic cytokine. Here we review recent developments in our understanding of the antiviral and immunoregulatory properties of IFN-alpha, the nature of the multisubunit IFN-alpha receptor, and the molecular mechanisms of signal transduction. Where available, we have included comparative data on recombinant alpha-IFNs derived from both naturally occurring and nonnaturally occurring synthetic genes. We also review clinical data and data on the side effects and antigenicity of different sources of recombinant alpha-IFNs in humans. These latter topics are of clinical interest, because they may potentially affect the efficacy of these various products. Hopefully, what is already known about IFN will prompt further exploration into the mechanism(s) of action of IFN-alpha and thus deliver new applications for this prototypic cytokine, whose full therapeutic potential is yet to be realized.
Antineoplastic Agents↗
Mutations in the double-stranded RNA-activated protein kinase insert region that uncouple catalysis from eIF2alpha binding.
The interferon-induced, double-stranded RNA (dsRNA)-activated protein kinase, PKR, inhibits protein synthesis via phosphorylation of the alpha subunit of the translation initiation factor eIF2. A kinase insert region N-terminal of PKR kinase subdomain V, which is conserved among eIF2alpha kinases, has been proposed to determine substrate specificity of these kinases. To investigate the function of this kinase insert region, selective PKR mutants were generated, and kinase activities and eIF2alpha affinities were analyzed in vitro. The in vivo function was investigated by growth inhibitory assays in yeast and translational assays in COS cells. Among the 13 mutations, 5 lost kinase activity and 3 exhibited less than 30% of wild-type eIF2alpha binding activity. The deletion of the conserved sequence (amino acids 362-370) resulted in a protein that had no kinase activity and only about 25% of wild-type eIF2alpha binding, suggesting that this sequence is not only required for PKR kinase activity but also is important for substrate interaction. It was determined that the hydrophobicity of the conserved sequence of PKR is required for kinase activity but is not crucial for eIF2alpha binding. The amino acid residue Glu-367 in the conserved motif was shown to be directly involved in substrate binding but was not important for kinase activity. These results suggest that the activation of PKR is not a prerequisite for its binding to the substrate and that the conserved motif in subdomain V contributes to the interaction of PKR and eIF2alpha.
Amino Acid Sequence↗
P53, bcl-2, and Bax expression in renal cell carcinoma.
OBJECTIVES: Renal cell carcinomas often show a high degree of resistance to chemotherapy and radiation despite expressing normal function of the protein p53. The loss of control of apoptosis may also contribute to progression and resistance to treatment modalities and can be attributed to an interaction between p53 and the apoptotic regulators bcl-2 and Bax. To determine whether the expression of p53, bcl-2, or Bax could be correlated with outcome, we analyzed the expression pattern of these proteins in renal cell tumor samples. METHODS: We examined 28 patients with clear cell renal cell carcinomas along with 7 patients with papillary renal cell carcinomas and 4 with renal oncocytomas. All renal cell carcinomas were clinically localized Stage pT2 with tumor size ranging from 4.0 to 10.3 cm (mean 6.23). Immunohistochemistry was performed on all samples and correlated with markers of outcome, including tumor grade, metastasis, recurrence, and overall survival rate. RESULTS: In all clear cell tumors, the detection level of p53 expression was below the sensitivity of the assay, consistent with the reported infrequent incidence of p53 mutations in renal cell cancers. bcl-2 expression showed a significant correlation (P = 0.018) with higher tumor grade but could not be significantly correlated with other parameters examined including tumor recurrence, metastasis, or survival rate. The expression of Bax could similarly be correlated with higher tumor grade but with none of the other parameters. CONCLUSIONS: At the present time, the combination of both tumor grade and stage represents the best prognostic markers available. Adjunctive use of bcl-2 and Bax staining currently plays a minimal role in helping to further stratify patients at high risk for disease progression or recurrence.
Adenoma, Oxyphilic↗
Progress in interferon and cytokine biology.
The 15th Annual Meeting of the International Society for Interferon and Cytokine Research was held on October 19-24, 1997, at the Sheraton San Diego Hotel and Marina. In this meeting Bryan Williams and Robert Silverman review the scientific highlights of the proceedings. Interferon regulatory factors (IRFs) were among the major topics discussed at this meeting. Fortunately, the scientific program of the meeting, organized by Tom Cesario (U.C., Irvine), was sufficiently interesting to hold cytokine biologists in darkened halls away from the splendid marina and sunny San Diego skies.
Animals↗
1H, 13C, 15N resonance assignment of the 20 kDa double stranded RNA binding domain of PKR.
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Binding Sites↗
Review of recent developments in the molecular characterization of recombinant alfa interferons on the 40th anniversary of the discovery of interferon.
Recombinant alfa interferons (IFN-alpha s) are approved worldwide for the treatment of a variety of cancers and diseases of virologic origin. A series of recent advances in the molecular characterization of recombinant IFN-alpha s have allowed the determination of the three-dimensional IFN-alpha 2b structure by high-resolution x-ray crystallography. We review here recent developments in our understanding of the molecular and physicochemical properties of recombinant IFN-alpha, including our current state of knowledge of the IFN-alpha gene family and the multiple species of human leukocyte IFN. Based on the reported three-dimensional structure of IFN-alpha 2b, we propose a molecular model for the IFN-alpha 2b receptor complex and predict models for the naturally occurring subtypes IFN-alpha 1 and IFN-alpha 8, as well as the synthetic, non-naturally occurring consensus IFN. Such models provide molecular insights into the mechanism of action of IFN-alpha.
Humans↗